Faithful initiation of the in vitro transcription of a cloned rDNA from Tetrahymena pyriformis.

A cloned rDNA fragment containing the transcriptional start point of Tetrahymena pyriformis was transcribed in vitro with a crude extract from homologous Tetrahymena cells. When a KpnI-HindIII fragment from the cloned rDNA plasmid was used as the template, the runoff transcription gave rise to two major RNA products about 490 and 460 nucleotides (nt) in length. An RNA of about 490 nt long was found to correspond to the expected transcript starting from the in vivo start point determined at our laboratory [Saiga et al., Nucl. Acids Res. 10 (1982) 4223-4235]. Precise size determination was performed using an RNA size marker prepared by hybridizing the template DNA with in vitro-capped 35S pre-rRNA followed by treatment with single-strand specific P1 nuclease. The size of the runoff transcript changed as predicted, according to downstream truncation points. The effects of alpha-amanitin and actinomycin D showed the transcription to be dependent on the added template DNA and to be catalyzed by form-I RNA polymerase. Possible reasons for the discrepancy between our determination of the size of the transcription product and that of Sutiphong et al. [Biochemistry 23 (1984) 6319-6326] are discussed.